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Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%

Author

Listed:
  • Joachim Goedhart

    (Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam)

  • David von Stetten

    (Structural Biology Group, European Synchrotron Radiation Facility)

  • Marjolaine Noirclerc-Savoye

    (CNRS, Institut de Biologie Structurale Jean-Pierre Ebel
    CEA, Institut de Biologie Structurale Jean-Pierre Ebel, 38027 Grenoble, France.
    Université Joseph Fourier, Institut de Biologie Structurale Jean-Pierre Ebel)

  • Mickaël Lelimousin

    (Structural Bioinformatics and Computational Biochemistry Unit, University of Oxford)

  • Linda Joosen

    (Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam)

  • Mark A. Hink

    (Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam)

  • Laura van Weeren

    (Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam)

  • Theodorus W.J. Gadella

    (Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam)

  • Antoine Royant

    (Structural Biology Group, European Synchrotron Radiation Facility
    CNRS, Institut de Biologie Structurale Jean-Pierre Ebel
    CEA, Institut de Biologie Structurale Jean-Pierre Ebel, 38027 Grenoble, France.
    Université Joseph Fourier, Institut de Biologie Structurale Jean-Pierre Ebel)

Abstract

Cyan variants of green fluorescent protein are widely used as donors in Förster resonance energy transfer experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into the brighter variants Super Cyan Fluorescent Protein 3A (SCFP3A) and mTurquoise, the latter exhibiting a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime. Here we combine X-ray crystallography and excited-state calculations to rationalize these stepwise improvements. The enhancement originates from stabilization of the seventh β-strand and the strengthening of the sole chromophore-stabilizing hydrogen bond. The structural analysis highlighted one suboptimal internal residue, which was subjected to saturation mutagenesis combined with fluorescence lifetime-based screening. This resulted in mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. Together, these properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Förster resonance energy transfer to a yellow fluorescent protein.

Suggested Citation

  • Joachim Goedhart & David von Stetten & Marjolaine Noirclerc-Savoye & Mickaël Lelimousin & Linda Joosen & Mark A. Hink & Laura van Weeren & Theodorus W.J. Gadella & Antoine Royant, 2012. "Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%," Nature Communications, Nature, vol. 3(1), pages 1-9, January.
    • Handle: RePEc:nat:natcom:v:3:y:2012:i:1:d:10.1038_ncomms1738
    DOI: 10.1038/ncomms1738
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    Cited by:

    1. Ariel A. Valiente-Gabioud & Inés Garteizgogeascoa Suñer & Agata Idziak & Arne Fabritius & Jérome Basquin & Julie Angibaud & U. Valentin Nägerl & Sumeet Pal Singh & Oliver Griesbeck, 2023. "Fluorescent sensors for imaging of interstitial calcium," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    2. Anna Rubinski & Noam E Ziv, 2015. "Remodeling and Tenacity of Inhibitory Synapses: Relationships with Network Activity and Neighboring Excitatory Synapses," PLOS Computational Biology, Public Library of Science, vol. 11(11), pages 1-29, November.
    3. Dorothy Koveal & Paul C. Rosen & Dylan J. Meyer & Carlos Manlio Díaz-García & Yongcheng Wang & Li-Heng Cai & Peter J. Chou & David A. Weitz & Gary Yellen, 2022. "A high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    4. Kirsty J Martin & Ewan J McGhee & Juliana P Schwarz & Martin Drysdale & Saskia M Brachmann & Volker Stucke & Owen J Sansom & Kurt I Anderson, 2018. "Accepting from the best donor; analysis of long-lifetime donor fluorescent protein pairings to optimise dynamic FLIM-based FRET experiments," PLOS ONE, Public Library of Science, vol. 13(1), pages 1-25, January.
    5. Stephanie Trauth & Ilka B Bischofs, 2014. "Ectopic Integration Vectors for Generating Fluorescent Promoter Fusions in Bacillus subtilis with Minimal Dark Noise," PLOS ONE, Public Library of Science, vol. 9(5), pages 1-9, May.

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